The second messengers cAMP and cGMP play important roles in mediating the biological effects of a wide variety of first messengers such as transducing a variety of extracellular signals, including hormones, light, and neurotransmitters. The intracellular levels of cAMP and cGMP are controlled by their rates of synthesis by cyclases and their rate of degradation by phosphodiestrases (PDEs).
Multiple families of PDEs have been identified (Beavo, J. A. (1995) Physiol. Rev. 75, 725-748; Soderling, S. H., Bayuga, S. J., Beavo, J. A. (1998) J. Biol. Chem. 273, 15553-15558; Fisher, D. A., Smith, J. F., Pillar, J. S., St. Denis, S. H., Cheng, J. B. (1998) J. Biol. Chem. 273, 15559-15564). Most of these families contain distinct genes, many of which are expressed in different tissues as alternative splice variants. Each PDE family has multiple isozymes and multiple splice variants displaying characteristic kinetic and regulatory properties, sequence homologies, and inhibitor profiles. Several lines of evidence have established an important role for PDEs in a wide variety of physiological processes. Genetic studies have indicated that different PDEs regulate such processes as learning and memory (Kauvar, L. M. (1982) J. Neurosci. 2, 1347-1358), development (Shaulsky, G., Escalante, R., Loomis, W. F. (1996) Proc. Natl. Acad. Sci. USA 93, 15260-15265), and visual signal transduction (McLaughlin, M. E., Sandberg, M. A., Berson, E. L., Dryja, T. P. (1993) Nat. Genet. 4, 130-134). Molecular and pharmacological studies have suggested that PDEs regulate such disparate functions as platelet aggregation (Dickinson, N. T., Jang, E. K., Hasalam, R. J. (1997) Biochem. J. 323, 371-377), aldosterone production, (MacFarland, R. T., Zelus, B. D., Beavo, J. A. (1991) J. Biol. Chem, 266, 136-142), insulin secretion (Zhao, A. Z., Zhao, H., Teague, J., Fujimoto, W., Beavo, J. A. (1997) Proc. Natl. Acad. Sci. USA 942, 3223-3228), and olfactory signal transduction (Yan, C., Zhao, A. Z., Bentley, J. K., Loughney, K., Ferguson, K., Beavo, J. A. (1995) Proc. Natl. Acad. Sci. USA 92, 9677-9681).
PDEs are typically composed of a catalytic domain (approximately 270 amino acids), an N-terminal regulatory domain responsible for binding cofactors, and, in some cases, a C-terminal domain of unknown function. A conserved motif, HDXXHXGXXN (SEQ ID NO.: 1), has been found in the catalytic domain of all PDEs. PDE families display roughly 35% amino acid homology within their catalytic domain. Isozymes within the same family typically display 75-95% homology in this region. Within a family, there is often greater than 60% homology outside the catalytic domain, whereas across different PDE families, there is little or no sequence similarity.
A variety of diseases have been suggested to result from decreased levels of cyclic nucleotides based on increased PDE activity. For example, altered PDE3 has been associated with cardiac disease (Smith, C. J., Huang, R., Sun, D., Ricketts, S., Hoegler, C., Ding, J. Z., Moggio, R. A., Hintze, T. H. (1997) Circulation 96, 3116-23). A form of diabetes insipidus in the mouse has been associated with increased PDE4 activity (Dousa, T. P. (1999) Kidney Int. 55, 29-62). Furthermore, defects in PDE6 have also been associated with retinal disease, such as retinal degeneration in mouse (Tsung, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. (1996) Science 272, 1026-9), autosomal recessive retinitis in humans (Baiget, M., Calaf, M., Valverde, D., del Rio, E., Reig, C., Carballo, M., Calvo, M. T., Gonzales-Duarte, R. (1998) Med. Clin. 111, 420-422), and rod/cone dysplasia in some dogs (Dekomien, G., Epplen, J. T. (2000) Anim. Genet. 31, 135-139).
PDEs have also been reported to effect cellular proliferation of a number of cell types and have been implicated in various types of cancer (Lemer, A., Kim, D. H., Lee, R. (2000) Leuk. Lymphoma 37, 39-51; Kim, D. H., Learner, A. (1998) Blood 92, 2484-94). Several of the PDEs, specifically, PDEs 3, 4 (Ekholm, D., Hemmer, B., Gao, G., Vergelli, M., Martin, R., and Manganiello, V. (1997) Journal Of Immunology 159, 1520-1529; Erdogan, S. and Houslay, M. D. (1997) Biochemical Journal 321,) and 7 (Giembycz, M. A., Corrigan, C. J., Seybold, J., Newton, R., and Barnes, P. J. (1996) Br J Pharmacol 118, 1945-58) have been reported to be expressed in human T cells. It is known that activation of CD4+ T cells requires stimulation of the CD3 receptor as well as costimulation of another receptor. Costimulation of the CD28 receptor leads to full activation of CD4+ T cells (Shahinian, A., Pfeffer, K., Lee, K. P., Kundig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. S., Thompson, C. B., and Mak, T. W. (1993) Science 261, 609-612).
It has been shown that PDE7A is upregulated in CD4+ T cells after CD3 and CD28 stimulation and that inhibition of PDE7A upregulation with an antisense oligo leads to inhibition of proliferation (Li, L., Yee, C., and Beavo, J. A. (1999) Science 283, 848-851). PDEs 3, 4 (Ekholm, D., Hemmer, B., Gao, G., Vergelli, M., Martin, R., and Manganiello, V. (1997) Journal Of Immunology 159, 1520-1529; Erdogan, S. and Houslay, M. D. (1997) Biochemical Journal 321) and 7 (Giembycz, M. A., Corrigan, C. J., Seybpld, J., Newton, R., and Barnes, P. J. (1996) Br J Pharmacol 118, 1945-58) have been reported to be expressed in human T cells.
Furthermore, PDE4 inhibitors have been found to be potent inhibitors of T cell proliferation (Manning, C. D., Burman, M., Christensen, S. B., Cieslinski, L. B., Essayan, D. M., Grous, M., Torphy, T. J., and Barnette, M. S. (1999): British Journal Of Pharmacology. Dec 128, 1393-1398).
Additional forms of PDEs have been described in yeast (Saccharomyces cerevisiae) (Nikawa J. et al., Mol Cell Biol 1987; 7: 3629-36), the slime mold Dictyostelium discoideum (Lacombe M. L. et al, J Biol Chem 1986; 261: 16811-7, Vibrio fisheri (Dunlap P. V. et al., J Bacteriol 1993; 175: 4615-24) and Candida albicans (Hoyer L. L. et al, Microbiology 1994; 140: 1533-42), that exhibit very little amino acid sequence identity with the previously described enzymes. These enzymes are currently designated as Class II PDEs, and likely have a different evolutionary origin, since, in contrast to all other eukaryotic PDEs, they have catalytic domains unlike those in mammalian Class I enzymes (Nikawa J. et al., Mol Cell Biol 1987; 7: 3629-36).
There is limited information about PDEs in trypanosomatids, a eukaryotic microorganism which causes the fatal human sleeping sickness (Vickerman, K. (1985) Br. Med-1. 41,105-114), as well as Nagana, a devastating disease of domestic animals in large parts of sub-Saharan Africa. Chemotherapy of human trypanosomiasis is in a dismal state (Seebeck, T. et al., (1999) Nova Act. Leopold. 78. 227-241). The cyclic nucleotide-specific PDEs may constitute a class of new drug targets.
cAMP signaling in trypanosomes is still largely unexplored (Naula, C. and Seebeck, T. (2000) Parasitol.Today 16, 35-38; Pays, E. et al., (1997) In: Trypanosomiasis and Leishmaniasis (Hide, G., Mottra, W. C., Coombs, G. H., and Holmes, P. H. eds.), 199-225). cAMP is involved in the regulation of differentiation of bloodstream form trypanosomes from the proliferative “long slender” forms to the insect-preadapted, non-proliferative “short stumpy” forms (Vassella, E. et al., (1997) J. Cell Sci. 110, 2661-2671). Several gene families for adenylyl cyclases have been identified in T. brucei (Naula, C., and Seebeck, T. (2000) Parasitol.Today 16, 35-38; Alexandre, S. et al., (1996) Mol Biochein. Parasitol. 77, 173-182; Alexandre, S. et al., (1990) Mol. Biochem. Parasitol. 43, 279-288). Even less is known about the trypanosomal phosphodiesterases. An early study demonstrated PDE activity in cell lysates of the bloodstream form T. brucei (Walter, R. D., and Opperdoes, F. R. (1982) Mol Biochem. Parasitol. 6, 287-295), and experiments with PDE inhibitors suggested that interference with PDE activity might affect cell differentiation (Vassella, E. et al., (1997) J. Cell Sci. 110, 2661-2671; Reed, S. L. et al., (1985) Infect. Immunol. 49, 844-847).